GSM7384515: 4Cseq_preiPSC_JMJD2C_Mut_hIDR1_R1; Mus musculus; OTHER
Approximately 5 million NS pre-iPSCs were harvested and crosslinked by formaldehyde. Resuspend cell pellet with 1ml low SDS Hi-C lysis buffer (0.1%SDS; 50mM HEPESKOH, pH7.5; 150mM NaCl; 1mM EDTA; 1% Triton X-100; 0.1% Sodium Deoxycholate) containing protease inhibitors, and incubate on ice for 30 min. Centrifuge at 2500g for 5 min at 4°C to discard supernatant. Then gently resuspend the cell pellet in 50 μl of 0.5% SDS and shake at 900 r.p.m. for exactly 10 minutes at 62°C on the thermomixer. 25 mL of the appropriate 10X New England Biolabs restriction enzyme buffer and 200 units of DpnII (NEB R0543) were added and the chromatin was incubated at 37°C degrees in a thermomixer at 500 RPM for four hours, 200 more units of DpnII (NEB R0543) was added and the reaction was incubated overnight at 37°C degrees in a thermomixer at 500 RPM. Inactivate DpnII incubating the sample for 20 min at 62°C while shaking at 500 rpm and then cool to room temperature. Proximity ligation was performed in a total of 1200 mL with 2000 units of T4 DNA ligase (NEB M020) for six hours at room temperature. After ligation samples were spun down for 5 minutes at 2500 rcf. and resuspended in 300 mL 10 mM Tris-HCl, 1% SDS and 0.5 mM NaCl with 1000 units of Proteinase K. Crosslinks were reversed by incubation overnight at 65°C. Samples were then phenol-chloroform extracted and ethanol precipitated and the second digestion was performed overnight in 450 mL with 50 units of restriction enzyme. BfaI (NEB R0568S) was used for Dppa5a. Samples were phenol-chloroform extracted and ethanol precipitated and the second ligation was performed in 14 mL total with 6700 units of T4 DNA ligase (NEB M020) at 16°C overnight. The sample was amplified unknown interacting sequences by inverse PCR with biotin-tagged oligos. Then, the library of sequency was prepared using the same strategy as the HiChIP